The molecular mechanisms behind DNA replication, an element essential to maintaining life, remain difficult for scientists to define or image.

But now a team of researchers has taken a major step to better understand the “replisome”—a block of proteins that act as molecular machinery to duplicate DNA. Their findings appear in the journal Nature Structural and Molecular Biology.

Until this research, scientists have not been able to produce a real structural image of a replisome at any resolution for any organism. By better understanding the structure and mechanisms of the replisome, scientists will be better equipped to understand what happens when DNA is miscopied, which can lead to cancer, or understand more about how cells multiply and develop into many cell types.

By using powerful electron microscopy techniques, Huilin Li, a professor in the biohemistry & cell biology department at Stony Brook University and a scientist at the US Department of Energy’s Brookhaven National Laboratory, and colleagues examined the shape of the replisome and piece by piece completed its structure.

What they saw brings new insight to how the molecular machinery functions and changes the long-held textbook view of what the replisome should look like.

“This work is a continuation of our long-standing research using electron microscopy to understand the mechanism of DNA replication, an essential function for every living cell,” says Li.

“These new images show the fully assembled and fully activated ‘helicase’ protein complex—which encircles and separates the two strands of the DNA double helix as it passes through a central pore in the structure—and how the helicase coordinates with the two ‘polymerase’ enzymes that duplicate each strand to copy the genome.”

Do textbooks have it wrong?

Li emphasizes that the finding and their observations challenge the textbook idea of helicase leading the way in the front to unwind the duplex DNA.

“All the textbook drawings and descriptions of how a replisome should look and work are based on biochemical and genetic studies,” Li explains, likening the situation to the famous parable of the three blind men trying to describe an elephant, each looking at only one part.

Those textbook drawings show the helicase moving along the DNA, separating the two strands of the double helix, with two polymerases located at the back where the DNA strand is split. In this configuration, the polymerases would add nucleotides (molecules containing the complementary A, T, G, and C bases of the genetic code) to the side-by-side split ends as they move out of the helicase to form two new complete double helix DNA strands.

To test these assumptions, Li’s group turned to the technique they had previously used to study individual components of the helicase, electron microscopy (EM). Jingchuan Sun, an EM expert in Li’s lab, studied samples of replisomes from baker’s yeast cells—a model for all nucleus-containing cells—prepared and provided by Roxana Georgescu in Michael O’Donnell’s research group at Rockefeller University.

“DNA replication is one of the most fundamental processes of life, so it is every biochemist’s dream to see what a replisome looks like,” Sun says.

Surprising results

The team’s first-ever images of an intact replisome revealed that only one of the polymerases is located at the back of the helicase. The other is onthe front side of the helicase, where the helicase first encounters the double-stranded helix. This means that while one of the two split DNA strands is acted on by the polymerase at the back end, the other has to thread itself back through or around the helicase to reach the front-side polymerase before having its new complementary strand assembled.

The scientists were so surprised by this finding that they asked another group at Rockefeller, led by Brian Chait, to perform additional structural studies using mass spectrometry. Yi Shi, a postdoctoral fellow in Chait’s group performed this work, which confirmed the electron-microscopy-based conclusion about the unexpected architecture of the replisome.

In terms of understanding what the implications of the true structure of the replisome are in molecular biology processes, the paper concludes, “Clearly, further studies will be required to understand the functional implications of the unexpected replisome architecture reported here.

The National Institutes of Health and by the Howard Hughes Medical Institute funded the work.